Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase

Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2β2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 A from the essential diferric-tyrosyl radical (Y122•) cofactor in β2. The Y122•-mediated oxidation of C439 in α2 occurs by a pathway (Y122 ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in α2) across the α/β interface. The absence of an α2β2 structure precludes insight into the location of Y356 and Y731 at the subunit interface. The proximity in the primary sequence of the conserved E350 to Y356 in β2 suggested its importance in catalysis and/or conformational gating. To study its function, pH–rate profiles of wild-type β2/α2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731, or both are reported in the presence of E350 or E350X (X = A, D, or Q) mutants. With E350, activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F2Y356 and F2Y731 are active and that radica...